Bacterial resistance to antibiotics has become a world-wide health crisis. Organisms that cause diarrhea, urinary tract infection, and sepsis are now resistant to many of the older antibiotics. The resulting health threat has prompted the search for structurally unique antibacterial agents with novel modes of action. Bicyclomycin is one such commercially available drug. We have discovered that the primary site for bicyclomycin function in Escherichia coli is the essential cellular protein, transcription termination factor Rho. In this proposal, we outline an integrated approach to further the understanding of the mechanisms of bicyclomycin expression. Research goals include (1) identifying the bicyclomycin binding domain in Rho and elucidating the role of key amino acids necessary for antibiotic activity, (2) elucidating the stoichiometry of the bicyclomycin-Rho complex and determining the regional disposition of the bicyclomycin binding pocket in relation to other functional domains within Rho, (3) determining the energetics of the drug-Rho binding process, (4) elucidating the mechanism of the bicyclomycin inhibition process, (5) correlating the in vitro and the in vivo bicyclomycin inhibitory pathways, and (6) determining the generality of the bicyclomycin inhibition pathway in microbial organisms. Chemical, biochemical, molecular biology, and biophysical methodologies will be used to meet these objectives. These include mass spectrometric analyses of bicyclomycin and ATP affinity-Rho complexes to identify the site and region of drug binding; generation and evaluation of random and site specific Rho mutations to identify amino acid residues required for drug function; use of BIAcore technology to determine the energetics for bicyclomycin-Rho interactions, fluorescence spectroscopy to monitor RNA-induced Rho processes, and X-ray crystallography to elucidate the structure of the protein-drug complex; use of bicyclomycin biomechanistic probes to determine the pathway for drug function; and development of an in vivo assay to correlate the in vitro and the in vivo mode of actions for bicyclomycin and bicyclomycin analogues. These investigations will provide the molecular basis to define novel pathways for bacterial control.